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Hum. Reprod. Advance Access originally published online on April 13, 2006
Human Reproduction 2006 21(8):2075-2083; doi:10.1093/humrep/del116
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© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Rapidly cooled human sperm: no evidence of intracellular ice formation

G.J. Morris1

Asymptote Ltd, St Johns Innovation Centre, Cambridge, UK

1 To whom correspondence should be addressed at: Asymptote Ltd, St Johns Innovation Centre, Cowley Road, Cambridge CB4 0WS, UK. E-mail: jmorris{at}asymptote.co.uk

BACKGROUND: The cellular damage that human spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice. However, no direct evidence of intracellular ice has been presented. Alternatively, the cell damage may be the result of an osmotic imbalance encountered during thawing. This article examines whether intracellular ice forms during rapid cooling or if an alternative mechanism is present. METHODS: In this study, human spermatozoa were cooled at a range of cooling rates from 0.3 to 3000°C/min. The ultrastructure of the samples was examined by cryo scanning electron microscopy and freeze substitution to determine whether intracellular ice formed during rapid cooling and to examine alternative mechanisms of cell injury during rapid cooling. RESULTS: No intracellular ice formation was detected at any cooling rate. Freeze substitution of cells that had been cooled at 3000°C/min and then slowly warmed showed that the cells had become plasmolysed and had evidence of membrane damage. CONCLUSIONS: Cell damage to human spermatozoa, at cooling rates of up to 3000°C/min, is not caused by intracellular ice formation. Spermatozoa that have been cooled at high rates are subjected to an osmotic shock when they are thawed.

Key words: freezing injury/intracellular ice/spermatozoa


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